Immunocytochemistry
Immunocytochemistry provides an accurate way of localizing antigens in situ in tissue sections or cell preparations using antibodies combined with a label that can be identified microscopically. The most frequently used labels are fluorescent compounds th
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34 Immunocytochemistry Susan Van Noorden 1. Introduction Immunocytochemistry provides an accurate way of localizing antigens in situ in tissue sections or cell preparations using antibodies combined with a label that can be identified microscopically. The most frequently used labels are fluorescent compounds that can be excited by light of specific wavelength, enzymes that can act on a specific substrate with a chromogen to give a colored end-product, and colloidal gold (for electron microscopy). Antibodies are applied to tissue preparations, often in a series of layers, one applied antibody acting as an antigen for the next (antispecies) antibody to build up the amount of label at the original site of reaction.
1.1. Conditions For any antigen localization, the antigen and the tissue must be fixed so that the antigen is insoluble and the tissue components are immobilized and protected from osmotic damage. Unfortunately, the “best” fixation for histology uses formalin, which crosslinks protein groups, followed by processing to paraffin sections; this fixation can sometimes damage antigenicity by altering the epitopes to which the antibody binds. For some antigens it is still necessary, therefore, to use fresh-frozen sections or cell preparations, postfixed in a precipitant fixative, such as acetone or alcohol. However, standard histological fixatives, such as formalin in its various combinations, can now usually be used in diagnostic histopathology, provided that antibodies have been selected during testing to react with fixed antigens, and with heat-mediated antigen retrieval methods an increasingly wide range of antibodies can be used on paraffin sections. From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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1.2. Diagnostic Applications Immunocytochemistry is now a routinely used diagnostic test for tumor type and innumerable other conditions in histo- and cytopathology, as well as in immunology, microbiology, and hematology. Immunocytochemistry is most useful in differential diagnosis, where a panel of antibodies to relevant antigens is used to distinguish among various options on material that cannot be diagnosed satisfactorily on standard histological preparations. Because this is a relatively expensive test, a blanket approach is not usually justified. Clinical history and preliminary examination of the histological preparations are important in selecting the panel to be used. The antibodies should have been extensively tested for specificity, sensitive methods should be used, and the appropriate negative and positive controls must always be carried out at the same time as the tests to ensure that the technique is in good working order and that the result is not caused by nonspecific reactions.
1.2.1. Antibody Screening During production of monoclonal antibodies (mAbs), rapid screening is required (Chapter 17). If the antibodies are to be used for immunocytochemistry, the scr
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